An unsolved, fundamental question concerning chromosomal translocations can be phrased as follows: "Do recurrent, non-random translocations occur between "recombinogenic" regions of the genome that are extraordinarily susceptible to breakage/religation events, or are the regions involved not particularly recombinogenic, but simply regions that lead to the production of oncogenic fusion proteins which confer a growth advantage to the cell." To help address this question, we developed an in vitro system in which we can produce chromosomal aberrations, through the use of the Isce-I restriction endonuclease, that do not provide a growth advantage to the cell. Using this system, we have learned that gross chromosomal rearrangements are a rare result of improper DNA repair of a double strand break, with a frequency roughly 1% that of small interstitial deletions. We are using this system to determine if gross chromosomal rearrangements will be more common if additional breaks are introduced, via genotoxic chemotherapy such as etoposide or bleomycin and/or VDJ recombination. Our preliminary results suggest that the use of etoposide and I-SceI does not lead to increased numbers of GCRs. However, the use of RAG1/RAG2 on a substrate that contains a VDJ recombination signal sequence has led to larger deletions, on the scale of 100s of kb. In addition, we are using this system to test whether purine/pyrimidine repeat regions are recombinogenic. We have expanded our studies to assay tumors derived from epithelial cells in addition to tumors derived from hematopoietic cells. Our results suggest that these also generate principally small interstitial deletions as opposed to GCRs. We also have begun experiments using a positive selection system (activation of a promotorless hygromycin resistance cassette) to determine if these would be more likely to develop GCRs. All of the hygromycin resistant clones we have recovered thus far following I-SceI transfection are due to plasmid "capture" events, where a portion of plasmid sequence containing SV40 regulatory regions has been captured at the I-SceI site. We have repeated these experiments in cells that are deficient for H2AX, a protein involved in normal DNA repair. Preliminary results indicate that some of these cells have undergone GCRs, including chromosomal translocations and inversions.